Molecular cloning of rat cardiac troponin I and analysis of troponin I isoform expression in developing rat heart

We have isolated and sequenced a cDNA encoding rat cardiac troponin I. The predicted amino acid sequence was highly identical with previously reported chemically derived amino acid sequences for rabbit and bovine cardiac troponin I. Clones for slow skeletal muscle troponin I were also obtained from neonatal rat cardiac ventricle by the polymerase chain reaction. The nucleotide sequences of these clones were determined to be more than 99% identical with a previously reported rat slow skeletal troponin I cDNA [Koppe et al. (1989) J. Biol. Chem. 264, 14327-14333]. The troponin I clones hybridized to RNA from the appropriate muscle from adult animals. However, RNA from fetal and neonatal rat heart also hybridized with the slow skeletal troponin I cDNA, demonstrating its expression in fetal and neonatal rat heart. Slow skeletal troponin I steady-state mRNA levels decreased with increasing age, but cardiac troponin I mRNA levels increased through fetal and early neonatal cardiac development. Thus, during fetal and neonatal development, slow skeletal and cardiac troponin I isoforms are coexpressed in the rat heart and regulated in opposite directions. The degree of primary sequence differences in these isoforms, especially at phosphorylation sites, may result in important functional differences in the neonatal myocardium.     

cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin I mRNAs

We have characterized the structure and expression of rodent mRNAs encoding the fast and slow skeletal muscle isoforms of the contractile regulatory protein, troponin I (TnIfast and TnIslow). TnIfast and TnIslow cDNA clones were isolated from mouse and rat muscle cDNA clone libraries and were used as isoform-specific probes in Northern blot and in situ hybridization studies. These studies showed that the TnIfast and TnIslow mRNAs are expressed in skeletal muscle, but not cardiac muscle or other tissues, and that they are differentially expressed in individual muscle fibers. Fiber typing on the basis of in situ hybridization analysis of TnI isoform mRNA content showed an excellent correlation with fiber type as assessed by myosin ATPase histochemistry. These results directly demonstrate that the differential expression of skeletal muscle TnI isoforms in the various classes of vertebrate striated muscle cells is based on gene regulatory mechanisms which control the abundances of specific TnI mRNAs in individual muscle cells. Both TnIfast and TnIslow mRNAs are expressed, at comparable levels, in differentiated cultures of rat L6 and mouse C2 muscle cell lines. Thus, although neuronal input has been shown to be an important factor in determining fast versus slow isoform-specific expression in skeletal muscle, both TnIfast and TnIslow genes can be expressed in muscle cells in the absence of nerve. Comparison of the deduced rodent TnI amino acid sequences with previously determined rabbit protein sequences showed that residues with potential fast/slow isoform-specific function are present in several discrete clusters, two of which are located near previously identified actin and troponin C binding sites.     

Developmental expression of troponin I isoforms in fetal human heart

We have used antibodies specific for troponin I proteins to examine human cardiac development and have detected a transiently expressed developmental isoform. This isoform is distinct from adult cardiac troponin I (TnIc) but is indistinguishable, on the basis of electrophoretic mobility and antibody reactivity, from the isoform found in slow skeletal muscle (TnIs). Furthermore, we show that mRNA for TnIs is present in fetal, but not adult, heart. Analysis of a developmental series of fetal samples indicates that there is a transition in expression from TnIs to TnIc which occurs between 20 weeks fetal and 9 months postnatal development.     

Troponin I switching in the developing heart

Monoclonal antibodies identify two distinct isoforms of troponin I in rat cardiac muscle, one predominant in the embryonic and fetal heart and one predominant in the adult heart. The two isoforms can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with apparent molecular weights of 27,000 and 31,500, respectively. The adult isoform is specifically recognized by a monoclonal antibody that is unreactive with the embryonic variant, while two other monoclonal antibodies recognize both isoforms. A monoclonal antibody to cardiac troponin T was used to isolate by affinity chromatography the troponin complex from adult and neonatal rat heart. Affinity purified troponin from neonatal heart was found to contain both the embryonic and adult isoforms of troponin I. Comparative immunoblotting analysis with different muscle tissues shows that embryonic troponin I is identical with respect to electrophoretic mobility and pattern of immunoreactivity to the major troponin I isoform found in adult slow skeletal muscle. Troponin I switching may be implicated in developmental changes involving Ca2+ and pH sensitivity of the contractile system and response to beta-adrenergic stimulation.     

Deletion of a Genomic Segment Containing the Cardiac Troponin I Gene Knocks Down Expression of the Slow Troponin T Gene and Impairs Fatigue Tolerance of Diaphragm Muscle

The loss of slow skeletal muscle troponin T (TnT) results in a recessive nemaline myopathy in the Amish featured with lethal respiratory failure. The genes encoding slow TnT and cardiac troponin I (TnI) are closely linked. Ex vivo promoter analysis suggested that the 5′-enhancer region of the slow TnT gene overlaps with the structure of the upstream cardiac TnI gene. Using transgenic expression of exogenous cardiac TnI to rescue the postnatal lethality of a mouse line in which the entire cardiac TnI gene was deleted, we investigated the effect of enhancer deletion on slow TnT gene expression in vivo and functional consequences. The levels of slow TnT mRNA and protein were significantly reduced in the diaphragm muscle of adult double transgenic mice. The slow TnT-deficient (ssTnT-KD) diaphragm muscle exhibited atrophy and decreased ratios of slow versus fast isoforms of TnT, TnI, and myosin. Consistent with the changes toward more fast myofilament contents, ssTnT-KD diaphragm muscle required stimulation at higher frequency for optimal tetanic force production. The ssTnT-KD diaphragm muscle also exhibited significantly reduced fatigue tolerance, showing faster and more declines of force with slower and less recovery from fatigue as compared with the wild type controls. The natural switch to more slow fiber contents during aging was partially blunted in the ssTnT-KD skeletal muscle. The data demonstrated a critical role of slow TnT in diaphragm function and in the pathogenesis and pathophysiology of Amish nemaline myopathy.     

Amino acid sequence of bovine cardiac troponin I

Troponin I (TnI) is the inhibitory subunit of troponin, the thin filament regulatory complex which confers calcium sensitivity to striated muscle actomyosin ATPase activity. We have determined the amino acid sequence of TnI from adult bovine cardiac muscle. This protein is a single polypeptide chain of 211 amino acids with an acetylated amino terminus, a calculated molecular weight of 23,975, and a net charge of +17 at neutral pH. There was no evidence for heterogeneity of the sequence. Comparison with other available TnI sequences shows an amino-terminal extension of 27-33 residues which is present in cardiac but not skeletal TnI. The remainder of the polypeptide is common to both cardiac and skeletal TnI. In the amino-terminal half of the common polypeptide, only 29% of the residues are invariant in all sequences. The carboxyl-terminal half (residues 124-210) is much more highly conserved, with 66% invariant residues. Bovine cardiac TnI and rabbit cardiac TnI are very similar in sequence: only 12 of 26 residues are identical in the amino-terminal segments, but the remaining residues of the proteins are 97% identical.     

A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA

We compare a recombinant cDNA plasmid (pAF81) complementary to a fetal skeletal muscle actin mRNA with a plasmid (pAM91) complementary to the actin mRNA expressed in adult skeletal muscle. The two mRNAs are significantly diverged in silent nucleotide positions; they are coexpressed in fetal skeletal muscle, and in differentiating muscle cell cultures their accumulation begins coordinately. The sequence of pAF81 shows that the amino acid sequence of mouse fetal skeletal muscle actin is almost identical to that of adult bovine cardiac actin. Hybridization of pAF81 to RNA from different mouse tissues shows that fetal skeletal muscle actin mRNA is very homologous or identical to fetal and adult cardiac actin mRNA. Only one gene homologous to pAF81 is detected on blots of restricted mouse DNA. We conclude that this gene must be expressed both in fetal skeletal muscle and in fetal heart. Whereas mRNA transcribed from this gene is the major actin mRNA species in adult heart, it is present in low amounts, if at all, in adult skeletal muscle.     

Role of troponin I isoform switching in determining the pH sensitivity of Ca(2+) regulation in developing rabbit cardiac muscle

Skinned muscle fibers prepared from fetal rabbit heart (28 days of gestation) showed a marked resistance to acidic pH in the Ca(2+) regulation of force generation, compared to the fibers prepared from adult heart. SDS-PAGE and immunoblot analysis showed that the slow skeletal troponin I was predominantly expressed in the fetal cardiac muscle, while the cardiac isoform was predominantly expressed in the adult cardiac muscle. Direct exchange of purified slow skeletal and cardiac troponin I isoforms into these skinned muscle fibers revealed that cardiac troponin I made the Ca(2+) regulation of contraction sensitive to acidic pH just as in the adult fibers, whereas slow skeletal troponin I made the Ca(2+) regulation of contraction resistant to acidic pH just as in the fetal fibers. These results demonstrate that the troponin I isoform switching accounts fully for the change in the pH dependence of Ca(2+) regulation of contraction in developmental cardiac muscle.     

Characterization of troponin T dilated cardiomyopathy mutations in the fetal troponin isoform

The major goal of this study was to elucidate how troponin T (TnT) dilated cardiomyopathy (DCM) mutations in fetal TnT and fetal troponin affect the functional properties of the fetal heart that lead to infantile cardiomyopathy. The DCM mutations R141W and DeltaK210 were created in the TnT1 isoform, the primary isoform of cardiac TnT in the embryonic heart. In addition to a different TnT isoform, a different troponin I (TnI) isoform, slow skeletal TnI (ssTnI), is the dominant isoform in the embryonic heart. In skinned fiber studies, TnT1-wild-type (WT)-treated fibers reconstituted with cardiac TnI.troponin C (TnC) or ssTnI.TnC significantly increased Ca(2+) sensitivity of force development when compared with TnT3-WT-treated fibers at both pH 7.0 and pH 6.5. Porcine cardiac fibers treated with TnT1 that contained the DCM mutations (R141W and DeltaK210), when reconstituted with either cardiac TnI.TnC or ssTnI.TnC, significantly decreased Ca(2+) sensitivity of force development compared with TnT1-WT at both pH values. The R141W mutation, which showed no significant change in the Ca(2+) sensitivity of force development in the TnT3 isoform, caused a significant decrease in the TnT1 isoform. The DeltaK210 mutation caused a greater decrease in Ca(2+) sensitivity and maximal isometric force development compared with the R141W mutation in both the fetal and adult TnT isoforms. When complexed with cardiac TnI.TnC or ssTnI.TnC, both TnT1 DCM mutations strongly decreased maximal actomyosin ATPase activity as compared with TnT1-WT. Our results suggest that a decrease in maximal actomyosin ATPase activity in conjunction with decreased Ca(2+) sensitivity of force development may cause a severe DCM phenotype in infants with the mutations.     

Cardiac troponin T in developing, regenerating and denervated rat skeletal muscle

Fetal rat skeletal muscles express a troponin T (TnT) isoform similar to the TnT isoform expressed in the embryonic heart with respect to electrophoretic mobility and immunoreactivity with cardiac TnT-specific monoclonal antibodies. Immunoblotting analyses reveal that both the embryonic and the adult isoforms of cardiac TnT are transiently expressed during the neonatal stages. In addition, other TnT species, different from both cardiac TnTs and from the TnT isoforms expressed in adult muscles, are present in skeletal muscles during the first two postnatal weeks. By immunocytochemistry, cardiac TnT is detectable at the somitic stage and throughout embryonic and fetal development, and disappears during the first weeks after birth, persisting exclusively in the bag fibers of the muscle spindles. Cardiac TnT is re-expressed in regenerating muscle fibers following a cold injury and in mature muscle fibers after denervation. Developmental regulation of this TnT variant is not coordinated with that of the embryonic myosin heavy chain with respect to timing of disappearance and cellular distribution. No obligatory correlation between the two proteins is likewise found in regenerating and denervated muscles.     

Myofilament incorporation determines the stoichiometry of troponin I in transgenic expression and the rescue of a null mutation

The highly organized contractile machinery in skeletal and cardiac muscles requires an assembly of myofilament proteins with stringent stoichiometry. To understand the maintenance of myofilament protein stoichiometry under dynamic protein synthesis and catabolism in muscle cells, we investigated the equilibrium of troponin I (TnI) in mouse cardiac muscle during developmental isoform switching and in under- and over-expression models. Compared with the course of developmental TnI isoform switching in normal hearts, the postnatal presence of slow skeletal muscle TnI lasted significantly longer in the hearts of cardiac TnI (cTnI) knockout (cTnI-KO) mice, in which the diminished synthesis was compensated by prolonging the life of myofilamental TnI. Transgenic postnatal expression of an N-terminal truncated cTnI (cTnI-ND) using α-myosin heavy chain promoter effectively rescued the lethality of cTnI-KO mice and shortened the postnatal presence of slow TnI in cardiac muscle. cTnI-KO mice rescued with different levels of cTnI-ND over-expression exhibited similar levels of myocardial TnI comparable to that in wild type hearts, demonstrating that excessive synthesis would not increase TnI stoichiometry in the myofilaments. Consistently, haploid under-expression of cTnI in heterozygote cTnI-KO mice was sufficient to sustain the normal level of myocardial cTnI, indicating that cTnI is synthesized in excess in wild type cardiomyocytes. Altogether, these observations suggest that under wide ranges of protein synthesis and turnover, myofilament incorporation determines the stoichiometry of troponin subunits in muscle cells.     

The highly conserved COOH terminus of troponin I forms a Ca2+-modulated allosteric domain in the troponin complex

The primary structure of the COOH-terminal region of troponin I (TnI) is highly conserved among the cardiac, slow, and fast skeletal muscle TnI isoforms and across species. Although no binding site for the other thin filament proteins is found at the COOH terminus of TnI, truncations of the last 19-23 amino acid residues reduce the activity of TnI in the inhibition of actomyosin ATPase and result in cardiac muscle malfunction. We have developed a specific monoclonal antibody (mAb), TnI-1, against the conserved COOH terminus of TnI. Using this mAb, isolation of the troponin complex by immunoaffinity chromatography from muscle homogenate and immunofluorescence microscopic staining of myofibrils indicate that the COOH terminus of TnI forms an exposed structure in the muscle thin filament. Binding of this mAb to the COOH terminus of cardiac TnI induced extensive conformational changes in the protein, suggesting an allosteric role of this region in the functional integrity of troponin. In the absence of Ca2+, the binding of troponin C and troponin T to TnI had very little effect on the conformation of the COOH terminus of TnI as indicated by the unaffected mAb affinity for the TnI-1 epitope. However, Ca2+ significantly increased the accessibility of the TnI-1 epitope on TnI in the presence of troponin C and troponin T. The results provide evidence that the COOH terminus is an essential structure in TnI and participates in the allosteric switch during Ca2+ activation of contraction.     

A pH-sensitive interaction of troponin I with troponin C coupled with strongly binding cross-bridges in cardiac myofilament activation

Slow skeletal muscle troponin I (ssTnI) expressed predominantly in perinatal heart confers a marked resistance to acidic pH on Ca(2+) regulation of cardiac muscle contraction. To explore the molecular mechanism underlying this phenomenon, we investigated the roles of TnI isoforms (ssTnI and cardiac TnI (cTnI)) in the thin filament activation by strongly binding cross-bridges, by exchanging troponin subunits in cardiac permeabilized muscle fibers. Fetal cardiac muscle showed a marked resistance to acidic pH in activation of the thin filament by strongly binding cross-bridges compared to adult muscle. Exchanging ssTnI into adult fibers altered the pH sensitivity from adult to fetal type, indicating that ssTnI also confers a marked resistance to acidic pH on the cross-bridge-induced thin filament activation. However, the adult fibers containing ssTnI or cTnI but lacking TnC showed no pH sensitivity. These findings provide the first evidence for the coupling between strongly binding cross-bridges and a pH-sensitive interaction of TnI with TnC in cardiac muscle contraction, as a molecular basis of the mechanism conferring the differential pH sensitivity on Ca(2+) regulation.     

Sarcomere thin filament regulatory isoforms. Evidence of a dominant effect of slow skeletal troponin I on cardiac contraction

Thin filament proteins tropomyosin (Tm), troponin T (TnT), and troponin I (TnI) form an allosteric regulatory complex that is required for normal cardiac contraction. Multiple isoforms of TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and 40-50% of native Tm or TnT were specifically replaced. The Ca(2+) sensitivity of tension development was markedly enhanced by TnI replacement but not by TnT or Tm isoform replacement. Titration of TnI replacement from >90% to <30% revealed a dominant functional effect of slow skeletal TnI to modulate regulation. Over this range of isoform replacement, TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction, and this identifies TnI as a potential target to modify contractile performance in normal and diseased myocardium.     

大鼠骨骼肌快肌肌钙蛋白T的同工型

骨骼肌快肌的收缩主要是由钙离子通过肌钙蛋白所调节控制。这些肌钙蛋白位于肌纤维之中。肌蛋白包括肌钙蛋白T、肌钙蛋白C、肌钙蛋白I。采用双向聚丙烯酰胺凝胶电泳和免疫学技术,对大鼠胚胎、新生大鼠和成年大鼠的骨能肌快肌肌钙蛋白T的同工型进行了研究。在成年大鼠的骨能肌快肌中,发现了10种肌钙蛋白T同工型。在大鼠胚胎和新生大鼠的骨能肌中,发现了7种肌钙蛋白T同工型。作为不同动物、不同发育阶段和不同组织发育的特殊标记,这些肌钙蛋白T同工型具有重要意义[动物学报49(3)：362—369,2003]。     

Identification of a region of fast skeletal troponin T required for stabilization of the coiled-coil formation with troponin I

We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca(2+)-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the alpha helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform.